There is increasing evidence of a worldwide epidemic of obesity and related conditions such as insulin resistance and diabetes. A major reason for the rapid rise in these conditions over the past several decades is related to increases in the caloric load and high fat content of typical diets. Understanding the contributions of dietary excess and specific macronutrients such as saturated fat to the development of insulin resistance and the underlying mechanisms by which this is achieved is therefore of critical importance for all segments of our population. We have recently demonstrated that a diet greatly enriched in saturated fatty acids (SFA) has a unique ability to induce a rapid (in d 24 hours) and profound whole body insulin resistance (~ 50% decline in glucose utilization) in humans. Importantly, initial biochemical and signal pathway characterization in tissues during the feeding of SFA-enriched diets indicates changes consistent with those typically present in chronic states of insulin resistance or diabetes. The first goal (Aim 1) of the current proposal, which is supported by both laboratory based studies and preliminary clinical data, will be to take advantage of this novel and physiologically relevant model to identify key signal pathways/mechanisms in cells and tissues responsible for dietary saturated fat induced insulin resistance. Primary mechanisms or pathways examined will include formation of bioactive lipid intermediates, generation of ER stress, and induction of mitochondrial dysfunction/reactive oxygen species in monocytes, skeletal muscle and adipose tissue as well as tissue and systemic inflammation. The rapid nature of this human model of insulin resistance will greatly facilitate identification of the early and therefore more proximal mechanisms underlying the metabolic changes of insulin resistance. Aim 1 will be achieved by conducting two clinical studies to determine the effects of saturated fat- enriched vs. healthy diet challenges on whole body insulin sensitivity and on the above noted mechanism pathways. Determining and comparing the effects of high SFA-enriched diets in those with normal and abnormal glucose metabolism, will also provide insight into the effect of baseline glucose tolerance/insulin resistance on the extent and mechanisms of responses to dietary fat challenge. Aim 2 will determine in a cross-over study the effects of dietary composition (monounsaturated fats vs. carbohydrates) on pathways of dietary induction of insulin resistance. In Aim 3, participants will be studied over increasing periods of high SFA intake, providing a comparison of changes in tissue composition and pathways over acute (1 meal), subacute (one 24-hour cycle of the SFA diet) and more chronic time frames (4 cycles of SFA diets). This aim will help determine the effects of repeated diet challenges on insulin resistance, gain insights into the temporal sequence of pathway events, identify initiating and secondary pathway changes, and clarify potential cross-talk between tissues. A major goal of these studies will be to demonstrate that this model can be a valuable clinical research tool to investigate mechanism underlying dietary induced insulin resistance in humans. As this is a rapid and physiologic model of insulin resistance, it provides the ability to rapidly accelerate the pace of investigatios into both effects and mechanisms of dietary fat (and potentially other macronutrients) on insulin resistance and potential therapies to arrest these processes.